Modulation of oligonucleotide CpG-mediated immune stimulation by positional modification of nucleosides

ABSTRACT

The invention provides methods for modulating the immune response caused by CpG dinucleotide-containing compounds. The methods according to the invention enables both decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/845,623, filed on Apr. 30, 2001, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/201,578, filed on May 1,2000. Each of the patent applications listed above is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the therapeutic use of oligonucleotides, bothin the antisense approach, and as immunostimulatory agents.

2. Summary of the Related Art

Oligonucleotides have become indispensable tools in modern molecularbiology, being used in a wide variety of techniques, ranging fromdiagnostic probing methods to PCR to antisense inhibition of geneexpression. This widespread use of oligonucleotides has led to anincreasing demand for rapid, inexpensive and efficient methods forsynthesizing oligonucleotides.

The synthesis of oligonucleotides for antisense and diagnosticapplications can now be routinely accomplished. See e.g., Methods inMolecular Biology, Vol 20: Protocols for Oligonucleotides and Analogspp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides andAnalogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991);and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech.6: 12 (1995); and Antisense Research and Applications (Crooke andLebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approachesincluded phosphodiester and phosphotriester chemistries. Khorana et al.,J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry foroligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179(1978), discloses phosphotriester chemistry for synthesis ofoligonucleotides and polynucleotides. These early approaches havelargely given way to the more efficient phosphoramidite andH-phosphonate approaches to synthesis. Beaucage and Caruthers,Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use ofdeoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawaland Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimizedsynthesis of oligonucleotides by the H-phosphonate approach.

Both of these modern approaches have been used to synthesizeoligonucleotides having a variety of modified internucleotide linkages.Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teachessynthesis of oligonucleotide methylphosphonates using phosphoramiditechemistry. Connolly et al., Biochemistry 23: 3443 (1984), disclosessynthesis of oligonucleotide phosphorothioates using phosphoramiditechemistry. Jager et al., Biochemistry 27: 7237 (1988), disclosessynthesis of oligonucleotide phosphoramidates using phosphoramiditechemistry. Agrawal et al., Proc. Anti. Acad. Sci. USA˜: 7079-7083(1988), discloses synthesis of oligonucleotide phosphoramidates andphosphorothioates using H-phosphonate chemistry.

More recently, several researchers have demonstrated the validity of theantisense approach to therapeutic treatment of disease. Crooke,Antisense Nucleic Acid Drug Dev. 8: vii-viii, discloses the successfulmarketing approval of a phosphorothioate oligonucleotide for thetreatment of human cytomegalovirus induced retinitis. Unfortunately, theuse of phosphorothioate oligonucleotides has become more complex thanoriginally expected. Certain effects caused by phosphorothioateoligonucleotides could not be explained by the expected antisensemechanism. For example, McIntyre et al., Antisense Res. Dev. 3: 309-322(1993) teaches that a “sense” phosphorothioate oligonucleotide causesspecific immune stimulation. This and other side effects havecomplicated the picture for phosphorothioate oligonucleotides.

On the other hand, the observation that phosphodiester andphosphorothioate oligonucleotides can induce immune stimulation hascreated interest in developing this side effect as a therapeutic tool.These efforts have focused on phosphorothioate oligonucleotidescontaining the dinucleotide CpG. Kuramoto et al., Jpn. J. Cancer Res.83: 1128-1131 (1992) teaches that phosphodiester oligonucleotidescontaining a palindrome that includes a CpG dinucleotide can induceinterferon-alpha and gamma synthesis and enhance natural killeractivity. Krieg et al., Nature 371: 546-549 (1995) discloses thatphosphorothioate CpG-containing oligonucleotides are immunostimulatory.Liang et al., J. Clin. Invest. 98: 1119-1129 (1996) discloses that sucholigonucleotides activate human B cells. Moldoveanu et al., Vaccine 16:1216-1224 (1998) teaches that CpG-containing phosphorothioateoligonucleotides enhance immune response against influenza virus.McCluskie and Davis, The Journal of Immunology 161: 4463-4466 (1998)teaches that CpG-containing oligonucleotides act as potent adjuvants,enhancing immune response against hepatitis B surface antigen.

These reports make clear that there is a need to be able to modulate theimmune response caused by CpG-containing oligonucleotides. Ideally, suchmodulation should include decreasing the immunostimulatory effect forantisense applications, as well as increasing the immunostimulatoryeffect for immunotherapy applications.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods for modulating the immune response causedby CpG dinucleotide-containing compounds. The methods according to theinvention enable both decreasing the immunostimulatory effect forantisense applications, as well as increasing the immunostimulatoryeffect for immunotherapy applications. Thus, the invention furtherprovides compounds having optimal levels of immunostimulatory effect foreither application and methods for making and using sucholigonucleotides.

The present inventor has surprisingly discovered that positionalmodification of CpG-containing oligonucleotides dramatically affectstheir immunostimulatory capabilities. In particular, 2′ or 3′ sugar orbase modifications of oligonucleotides, or introduction of a modifiedinternucleoside linkage, at particular positions 5′ or 3′, including 5′or 3′ end modifications, to the CpG dinucleotide either enhances orreduces their immunostimulatory effect in a reproducible and predictablemanner.

In a first aspect, the invention provides a method for modulating theimmunostimulatory effect of a CpG dinucleotide containing compound byintroducing an immunomodulatory moiety at a position either 5′ to or 3′to the CpG dinucleotide.

In a second aspect, the invention provides compounds having increased orreduced immunostimulatory effect, the compounds comprising a CpGdinucleotide and an immunomodulatory moiety, wherein the increased orreduced immunomodulatory effect is relative to a similar compoundlacking the immunomodulatory moiety.

In a third aspect, the invention provides a method for obtaining anantisense-specific reduction in the expression of a gene in a mammal,including a human, the method comprising administering to the mammal anoligonucleotide that is complementary to the gene and which comprises aCpG dinucleotide and an immunomodulatory moiety, wherein theoligonucleotide has less immunostimulatory effect than a similaroligonucleotide lacking the immunomodulatory moiety.

In a fourth aspect, the invention provides a method for inducing animmune response in a mammal, including a human, the method comprisingadministering to the mammal a compound comprising a CpG dinucleotide andan immunomodulatory moiety, wherein the compound has greaterimmunostimulatory effect than a similar compound lacking theimmunomodulatory moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows preferred embodiments of immunomodulatory moietiesaccording to the invention. Note that the Figures use X3X4 for the 3′side and X1X2 for the 5′ side. This use is illustrative for this figureonly and should not be used to interpret the claims, which use the Y andX designations taught in this specification.

FIG. 2 (SEQ ID NOs 1-3, respectively, in order of appearance) shows amodified compound according to the invention and results of spleenassays using this compound.

FIG. 3 (SEQ ID NOs 4-6, respectively, in order of appearance) showsanother modified compound according to the invention and results ofspleen assays using this compound.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the therapeutic use of oligonucleotides, bothin the antisense approach, and as immunostimulatory agents. The patentsand publications cited herein reflect the level of knowledge in thefield and are hereby incorporated by reference in their entirety. In theevent of conflict between any teaching of any reference cited herein andthe present specification, the latter shall prevail, for purposes of theinvention.

The invention provides methods for modulating the immune response causedby CpG dinucleotide-containing compounds. The methods according to theinvention enable both decreasing the immunostimulatory effect forantisense applications, as well as increasing the immunostimulatoryeffect for immunotherapy applications. Thus, the invention furtherprovides oligonucleotides having optimal levels of immunostimulatoryeffect for either application and methods for making and using sucholigonucleotides.

The present inventor has surprisingly discovered that positionalmodification of CpG-containing oligonucleotides dramatically affectstheir immunostimulatory capabilities. In particular, 2′ or 3′ sugar orbase modifications of oligonucleotides, or introduction of a modifiedinternucleoside linkage, at particular positions 5′ or 3′ to the CpGdinucleotide, including 5′ or 3′ end modifications, or combinationsthereof, either enhances or reduces their immunostimulatory effect in areproducible and predictable manner.

In a first aspect, the invention provides a method for modulating theimmunostimulatory effect of a CpG dinucleotide containing compound byintroducing an immunomodulatory moiety at a position either 5′ to or 3′to the CpG dinucleotide. Preferred compounds according to this aspect ofthe invention generally include additional oligonucleotide sequences.

In certain preferred embodiments the method is used to make anoligonucleotide that is complementary to a gene or gene transcript. Incertain preferred embodiments, the oligonucleotide has antisenseactivity. In some preferred embodiments, only one immunomodulatorymoiety is introduced into the oligonucleotide for each CpG dinucleotidepresent in the oligonucleotide. In some preferred embodiments, only oneimmunomodulatory moiety is introduced into the oligonucleotide.

In certain preferred embodiments, the oligonucleotide made according tothis aspect of the invention does not have antisense activity and/or isnot complementary to a gene.

As herein, the term “complementary” means having the ability tohybridize to a genomic region, a gene, or an RNA transcript thereofunder physiological conditions. Such hybridization is ordinarily theresult of base-specific hydrogen bonding between complementary strands,preferably to form Watson-Crick or Hoogsteen base pairs, although othermodes of hydrogen bonding, as well as base stacking can also lead tohybridization. As a practical matter, such hybridization can be inferredfrom the observation of specific gene expression inhibition.

As used herein, “antisense activity” means that the oligonucleotide,when introduced into a cell or an animal, causes a reduction in theexpression of the gene to which it is complementary.

The method according to this aspect of the invention can be convenientlycarried out using any of the well-known synthesis techniques by simplyusing an appropriate immunomodulatory moiety monomer synthon in thesynthesis process in a cycle following, immediately or otherwise theincorporation of the CpG dinucleotide. Preferred monomers includephosphoramidites, phosphotriesters and H-phosphonates.

For purposes of the invention, “introducing an immunomodulatory moietyinto position Y2” simply means synthesizing an oligonucleotide that hasan immunomodulatory moiety at such a position, with reference to thefollowing structure: 5′-Yn . . . Y6-Y5-Y4-Y3-Y2-Y1-CG-X1-X2-X3-X4-X5-X6-X7-X8-X9 . . . Xm-3′,

wherein C is cytosine, G is guanosine, a substituted guanosine,including inosine and 7-deazaguanosine, and each X and Y isindependently a nucleoside or an immunomodulatory moiety, and n is anumber from −6 to +20, and m is a number from −9 to +20.

Procedures for synthesis of oligonucleotides are well known in the art.

In a second aspect, the invention provides compounds having increased orreduced immunostimulatory effect, the compounds comprising a CpGdinucleotide and an immunomodulatory moiety, wherein the increased orreduced immunomodulatory effect is relative to a similar compoundlacking the immunomodulatory moiety. Preferred compounds according tothis aspect of the invention generally include additionaloligonucleotide sequences. Preferably, such oligonucleotide sequenceswill have from about 6 to about 50 nucleotides, most preferably fromabout 12 to about 35 nucleotides.

Certain preferred compounds according to the invention have thestructure: 5′-Yn . . . Y6-Y5-Y4-Y3-Y2-Y1-CG--X1-X2-X3-X4-X5- X6-X7-X8-X9. . . Xm-3′,

wherein C is cytosine, G is guanosine, a substituted guanosine,including inosine and 7-deazaguanosine, and each X and Y isindependently a nucleoside or an immunomodulatory moiety, and n is anumber from −6 to +20, and m is a number from −9 to +20.

In particularly preferred embodiments, the base sequence that ismodified to provide the compound is 5′-CTATCTGACGTTCTCTGT-3′ (SEQ IDNO 1) or 5′-CCTACTAGCGTTCTCATC-3′ (SEQ ID NO 4)Preferred immunomodulatory moieties include one or more abasicnucleoside, 1,3-propanediol linker (substituted or unsubstituted),and/or modified base-containing nucleosides, including nitropyrrole,nitroindole, deoxyuridine, inosine, isoguanosine, 2-aminopurine,nebularine, 7-deazaguanosine, 4-thiodeoxyuridine, 4-thiothymidine,d-isoguanosine, d-iso-5-methylcytosine, P-base, and 3′-3′ linkage. As ageneral rule, introduction of an immunomodulatory moiety at position Y6,Y5, Y4, or Y3, or a combination thereof, increases the immunostimulatoryeffect of the oligonucleotide. Generally, introduction of animmunomodulatory moiety at position Y2 maintains immunostimulatoryeffect. Generally, introduction of an immunomodulatory moiety atposition Y1 maintains or reduces immunostimulatory effect. Generally,introduction of an immunomodulatory moiety at position C abolishesimmunostimulatory effect. Generally, introduction of an immunomodulatorymoiety at position G abolishes immunostimulatory effect, except for7-deazaguanosine, which maintains immunostimulatory effect. Generally,introduction of an immunomodulatory moiety at position X1 maintains orreduces immunostimulatory effect. Generally, introduction of animmunomodulatory moiety at position X2 has little impact onimmunostimulatory effect. Generally, introduction of an immunomodulatorymoiety at position X3 maintains or increases immunostimulatory effect.Generally, introduction of an immunomodulatory moiety at position X4,X5, X6, X7-Xm, or any combination thereof, increases immunostimulatoryeffect.

Certain preferred oligonucleotides according to this aspect of theinvention are complementary to a gene or gene transcript. Morepreferably, such oligonucleotides have antisense activity. In somepreferred embodiments, the oligonucleotide has only one immunomodulatorymoiety for each CpG dinucleotide present in the oligonucleotide. In somepreferred embodiments, the oligonucleotide has only one immunomodulatorymoiety. In other preferred embodiments, the compounds according to thisaspect of the invention do not have antisense activity and/or are notcomplementary to a gene.

In a third aspect, the invention provides a method for obtaining anantisense-specific reduction in the expression of a gene in a mammal,including a human, the method comprising administering to the mammal anoligonucleotide that is complementary to the gene and which comprises aCpG dinucleotide and an immunomodulatory moiety, wherein theoligonucleotide has less immunostimulatory effect than a similaroligonucleotide lacking the immunomodulatory moiety.

In some preferred embodiments, the oligonucleotide has only oneimmunomodulatory moiety for each CpG dinucleotide present in theoligonucleotide. In some preferred embodiments, the oligonucleotide hasonly one immunomodulatory moiety.

In the methods according to this aspect of the invention, preferably,administration of antisense oligonucleotides should be parenteral, oral,sublingual, transdermal, topical, intranasal, intratracheal, orintrarectal. Administration of the therapeutic compositions can becarried out using known procedures at dosages and for periods of timeeffective to reduce symptoms or surrogate markers of the disease. Whenadministered systemically, the therapeutic composition is preferablyadministered at a sufficient dosage to attain a blood level ofoligonucleotide from about 0.001 micromolar to about 10 micromolar. Forlocalized administration, much lower concentrations than this may beeffective, and much higher concentrations may be tolerated. Preferably,a total dosage of oligonucleotide will range from about 0.1 mgoligonucleotide per patient per day to about 200 mg oligonucleotide perkg body weight per day. It may be desirable to administersimultaneously, or sequentially a therapeutically effective amount ofone or more of the therapeutic compositions of the invention to anindividual as a single treatment episode. In a preferred embodiment,after the composition of matter is administered, one or more measurementis taken of biological effects selected from the group consisting ofcomplement activation, mitogenesis and inhibition of thrombin dotformation.

The method according to this aspect of the invention is useful in animalmodels of disease or gene expression, and is further useful for thetherapeutic treatment of human or animal disease.

In a fourth aspect, the invention provides a method for inducing animmune response in a mammal, including a human, the method comprisingadministering to the mammal a compound comprising a CpG dinucleotide andan immunomodulatory moiety, wherein the compound has greaterimmunostimulatory effect than a similar compound lacking theimmunomodulatory moiety.

In the methods according to this aspect of the invention, preferably,administration of compounds should be parenteral, oral, sublingual,transdermal, topical, intranasal, intratracheal, or intrarectal.Administration of the therapeutic compositions can be carried out usingknown procedures at dosages and for periods of time effective to reducesymptoms or surrogate markers of the disease. When administeredsystemically, the therapeutic composition is preferably administered ata sufficient dosage to attain a blood level of oligonucleotide fromabout 0.001 micromolar to about 10 micromolar. For localizedadministration, much lower concentrations than this may be effective,and much higher concentrations may be tolerated. Preferably, a totaldosage of oligonucleotide will range from about 0.1 mg oligonucleotideper patient per day to about 200 mg oligonucleotide per kg body weightper day. It may be desirable to administer simultaneously, orsequentially a therapeutically effective amount of one or more of thetherapeutic compositions of the invention to an individual as a singletreatment episode. In a preferred embodiment, after the composition ofmatter is administered, one or more measurement is taken of biologicaleffects selected from the group consisting of complement activation,mitogenesis and inhibition of thrombin clot formation.

In certain preferred embodiments, compounds according to the inventionare administered in combination with vaccines, antibodies, cytotoxics,antisense oligonucleotides, gene therapy vectors, DNA vaccines and/oradjuvants to enhance the specificity or magnitude of the immuneresponse. Either the compound or the vaccine, or both may optionally belinked to an immunogenic protein, such as keyhole limpet hemocyanin,cholera toxin B subunit, or any other immunogenic carrier protein. Anyof the plethora of adjuvants may be used, including, without limitation,Freund's complete adjuvant. For purposes of this aspect “in combinationwith” means in the course of treating the same ‘disease in the samepatient, and includes administering the oligonucleotide and/or thevaccine and/or the adjuvant in any order, including simultaneousadministration, as well as temporally spaced order of up to several daysapart. Such combination treatment may also include more than a singleadministration of the oligonucleotide, and/or independently the vaccine,and/or independently the adjuvant. The administration of theoligonucleotide and/or vaccine and/or adjuvant may be by the same ordifferent routes.

The method according to this aspect of the invention is useful for modelstudies of the immune system, and is further useful for the therapeutictreatment of human or animal disease.

For purposes of all aspects of the invention, the term “oligonucleotide”includes polymers of two or more deoxyribonucleotides, or any modifiednucleoside, including 2′-halo-nucleosides, 2′ or 3′ substituted, 2′ or3′-O-substituted ribonucleosides, deazanucleosides or any combinationthereof. Such monomers may be coupled to each other by any of thenumerous known internucleoside linkages. In certain preferredembodiments, these internucleoside linkages may be phosphodiester,phosphotriester, phosphorothioate, or phosphoramidate linkages, 2′-5′linkages of any of the forgoing, or combinations thereof. The termoligonucleotide also encompasses such polymers having chemicallymodified bases or sugars and/or having additional substituents,including without limitation lipophilic groups, intercalating agents,diamines and adamantane. The term oligonucleotide also encompasses PNA,LNA and oligonucleotides comprising non-pentose sugar (e.g. hexose)backbones or backbone sections. For purposes of the invention the terms“2′-O-substituted” and “3′-O-substituted” mean (respectively)substitution of the 2′ (or 3′) position of the pentose moiety with ahalogen (preferably Cl, Br, or F), or an —O-lower alkyl group containing1-6 saturated or unsaturated carbon atoms, or with a-O-aryl or allylgroup having 2-6 carbon atoms, wherein such alkyl, aryl or allyl groupmay be unsubstituted or may be substituted, e.g., with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl, or amino groups; or such 2′ substitution may be with ahydroxy group (to produce a ribonucleoside), an amino or a halo group,but not with a 2′ (or 3′) H group. For purposes of all aspects of theinvention, the terms “CpG” or “CpG dinucleotide” means the dinucleotide5′-deoxycytidine-deoxyguanidine or deoxyguanidine analog-3′, wherein pis an internucleotide linkage, and wherein the sugar backbone of thedinucleotide may be ribose, deoxyribose, or 2′ substituted ribose, orcombinations thereof. In preferred embodiments of the first threeaspects of the invention, p is selected from phosphodiester,phosphorothioate, alkylphosphonate, phosphotriester, stereospecific (Rpor Sp) phosphorothioate or alkylphosphonate, and 2′-5′ covalent linkagesof any of the above. The non-phosphodiester, non-phosphorothioateembodiments will further reduce immunostimulatory effects. In preferredembodiments of the last three aspects of the invention, p is selectedfrom phosphodiester, phosphorothioate and phosphorodithioate.

The following examples are intended to further illustrate certainpreferred embodiments of the invention, and are not intended to limitthe scope of the invention.

EXAMPLE 1 Modulation of Immunostimulatory Effect In Vitro

To study the impact of site of chemical modification of PS-oligoscontaining CpG motif, we chose two oligonucleotides. Oligo 1 and Oligo2, each of which contains one CpG motif. To evaluate theimmunostimulatory activity of oligonucleotides in the present study, wewill use a mouse spleen cell proliferation assay.

Mouse spleen lymphocytes are cultured with oligonucleotides atconcentration of 0.1, 1, and 10 μg/mL. Oligo 1 and Oligo 2 will induce adose dependent effect on cell proliferation. At 0.1 μg/mL, theproliferation index will increase. Substitution of 5′-flankingdeoxynucleoside (Y1) of CpG motif of Oligo 1 or Oligo 2 with animmunomodulatory moiety according to the invention will result incomplete suppression of cell proliferation at all concentrations used(FIG. 1). At 0.1 μg/mL, cell proliferation index will be similar tomedium alone. Substitution of the 3′-flanking deoxynucleoside (X1) ofCpG motif of Oligo 1 or Oligo 2 with 2′-OMe will not have such an impacton cell proliferation, but may reduce it slightly. Similar substitutionsare made in Oligo 1 or Oligo 2 in the 3′-flanking region to CpG motif.Oligos are synthesized in which a deoxynucleoside is substituted with animmunomodulatory moiety according to the invention at position X3, X4,X5 or X6. The proliferation index of these oligos will increase.

EXAMPLE 2 Effect of Immunomodulatory Moieties on Spleen Weight

After observing that above substitutions in modulates itsimmunostimulatory activity based on cell culture assay, we administeroligonucleotides listed in Table 1 intraperitonealy to mice and measurethe spleen weights to confirm that the substitutions have similareffects in vivo. Administration of Oligo 1 or Oligo 2 will causesubstantial increase in spleen weight. Substitution of a deoxynucleotideaway from CpG motif towards 5′-end, positions Y6, Y5, Y4 or Y3 willcause progressive increase in spleen weights confirming an increase intheir immunostimulatory activity. Substitutions of a deoxynucleosidetoward the 3′-end of the CpG motif, in general, will cause lesssignificant increase in spleen weight. Data are shown in FIGS. 2 and 3.

EXAMPLE 3 Synthesis of Oligonucleotides Containing ImmunomodulatoryMoieties

Oligonucleotides are synthesized on 1 micromolar scale using anautomated DNA synthesizer (Expedite 8909, PerSeptive Biosystems, FosterCity, Calif.). Standard deoxynucleoside phosphoramidites are obtainedfrom PerSeptive Biosystems. 1′,2′-dideoxyribose phosphoramidite,propyl-1-phosphoramidite, 2′-deoxy-5-nitroindole-ribofuranosylphosphoramidite, deoxyuridine phosphoramidite, dP phosphoramidite,d-2-aminopurine phosphoramidite, d-nebularine phosphoramidite andd-7-deazaguanine phosphoramidite are obtained from Glen Research(Sterling, Va.). Deoxyinosine phosphoramidite is obtained from ChemGenes(Ashland, Mass.). Normal coupling cycles are used for allphosphoramidites. Beaucage reagent is used as an oxidant to obtainphosphorothioate modification. After synthesis, oligonucleotides aredeprotected by incubating CPG-bound oligonucleotide with concentratedammonium hydroxide solution for 1.5-2 hours at room temperature and thenincubating the ammonium hydroxide supernatant for 12 hours at 55 degreesC. The ammonium hydroxide solution is evaporated to dryness in aspeed-vac and 5′-DMTr-oligonucleotides are purified by HPLC on a C18reverse-phase matrix using a solvent system of 0.1 M ammonium acetateand 1:5 ratio 0.1 M ammonium acetate in acetonitrile. Then theoligonucleotides are treated with 80% acetic acid to remove the DMTrgroup, converted to sodium form and desalted by dialysis againstdistilled water. Oligonucleotides are lyophilized and redissolved inwater. Characterization is achieved by denaturing PAGE and MALDI-TOFmass spectrometry.

1.-22. (canceled)
 23. An immunostimulatory oligonucleotide having thestructure 5′-Yn . . . Y6-Y5-Y4-Y3-Y2-Y1-CG-X1-X2-X3-X4-X5- X6-X7-X8-X9 .. . Xm-3′,

wherein C is cytosine, G is 7-deazaguanosine, and each X and Y isindependently a nucleoside or an immunomodulatory moiety, and n is anumber from −6 to +20, and m is a number from −9 to +20.